Jamille,

I think you will have quite the challenge ahead of you, but not impossible. I have run hundreds of ELISA's, but honestly do not have experience in developing an original ELISA kit. The key to your success will be developing a quality antibody that binds your target it its native state (as opposed to denatured, which is optimal for Western blots). Once you have this, the rest is simply attaching said antibody to a plate, adding experimental samples, washing, secondary, developing, etc.

I have attached a comprehensive discussion on ELISA's by Thermo Scientific, which I think will be of help in your process of developing an original ELISA. Good luck.

~Adam

I would start with a literature search. The obvious (classic) way of doing this is to use bacterial lysates to coat the plates, block, incubate diluted serum in blocking solutions and the detect the bound antibodies with a suitable conjugate. A lot of individual details relate to the conditions that you have in your lab.

The problem of using bacterial lysates, however, is that they contain biomolecules (proteins, LPS, lipids etc.) that have a high similarity to other bacteria and lead to cross-reactivity. So, the clinical specificity of the test is not very high. Typically, today one would use recombinant proteins, or even fragments, because they make possible to detect only specific antibodies. Or you employ extraction protocols to end-up with specific fractions. Often it is a good idea to enrich surface proteins but it is not always sufficient That's why you need to do the literature search in the first place. Hope to find ideas about the antigens to employ.

First you should have a methodology paper, coating antigen (the mentioned bacteria or its products),polysorb plates, blocking material like bovine serum albumine, phosphate buffer saline, sodium azide, tween 20, the tested serum samples, specific conjugate and its specific substrate in addition to H2so4 or NaoH as a stopping solution. Then you estimate your calculations according to the methodology paper for example amount of antigen per well, composition of blocking and washing solutions, dilution factor of conjugate..................etc then after write ELISA steps in a paper and take it as a schedual for you and start work.

Thanks everyone, this gives me some great idea's on how to start. Swati, I want to develop an ELISA assay for detecting Pasturella in serum and was thinking that the best way to detect it would be by detecting it's antibodies.

Try coating/immobilizing Pasturella on a plate or bead. Incubate the serum sample, wash and add a labeled anti-human IgG for detection.

I fully agree with the suggestions and comments of Lars Komorowski .Others' suggestions are also relevant. Methodology is not new. Follow the suggestions of Lars Komorowski to find specific antibody against specific antigen, you will be successful in detection of the organism.

Indeed, a good literature research is important to find standard protocols. An interesting site with much information can be found here: http://www.abdserotec.com/resources/elisa-technical-resources-and-troubleshooting/helpful-elisa-hints.html (obviously, there are plenty of them around the internet).

Depending on the type of ELISA you would like to perform, you will have to optimize several crucial parameters: antibody (or antigen) coating (PBS, carbonate buffer, depending on the IEP of the agent which must be coated), type of blocking agent (BSA, gelatin, commercial blocking agents), optimal antibody / antigen concentration (and incubation times), selection of primary and secondary antibodies, ... For detection, you can use biotin-coated antibodies in combination with a streptavidin-HRP complex or second antibodies which are directly conjugated to HRP, chemiluminiscent or colorimetric detection ... Quite a lot of work, but within a couple of months, you must be able to have a workable "draft" version of your ELISA. Good luck!

PS Use 96-well plates with high protein binding capacity (e.g. Nunc Maxisorp plates)

PS2 washing steps are crucial to avoid high background readings

You need a couple of things:

1. an antigen on the solid phase and different ways to get this

1.1. whole bacteria (difficult to get them stable at a solid phase

1.2. lysates: major disadvantage: you will get large proteins and DNA much more stable than the smaller surface proteins or even carbohydrates

1.3. indirect binding: here you need a existing specific antibody (polyclonal or monoclonal, whatever is available) which have to bind previously at the solid phase. this antibody will bind the the specific antigen from the bacterial lysate. The disadvantage of this method is, that you have only the distinct antigen (indirectly bound). It's could be a disadvantage but it's could be also an advantage if you take into account, that many antigens which are responsible for unspecific or cross reactions.

1.4. Fractions or even purified bacterial antigens (if available via catalog)

1.4.1. proteins can be bound easily in 0.1 ml/L carbonate buffer, pH 9.6

1.4.2 mixed antigens including carbohydrates will bind indirectly via methylated BSA (precoating 5 µg/mL in carbonate buffer)

1.4.3 or any other tricks like biotinylation etc. are allowed

2. positive control sera/plasma

2.1. from infected patients / animals

2.2. from immunized animals / volunteers / patients

3. negative control sera / plasma

it's can become a problem if the bacteria are ubiquitous. Absorption can be helpful here. Inactivated bacteria will be incubated with the serum at 4 °C overnight and removed with the bound antibody by high speed centrifugation

4. an conjugate

4.1 if you would like to work species specific an anti-species Ig-enzyme labelled (HRP is the best)

4.1.1. if you would like to look for different immunoglobulin classes -> a class specific anti-Gamma/µ/alpha, ...-HRP

4..1.2 if not, you can use an anti-L (Kappa/Lambda)-HRP

4.2 if you would like to build an ELISA working for more than one species HRP-laballed Protein-A / Protein-G are available (see here: http://www.seramun.com/index.html)

5. buffer

5.1 coating buffer: 0.1 mol/L carbonate pH 9.6

5.2 washing buffer: PBS 0.3 mol/L NaCl (the increase of the NaCl concentration will decrease unspecific reactions) + 0.1% v/v Tween 20

5.3 incubation buffer: washing buffer + 1 ... 3% inert protein (BSA, HSA, Gelatine, ...). Add some phenol red to see much more easier where you are currently pipetting.

5.4. substrate (TMB, ready to use ... ). see here: http://www.seramun.com/index.html

5.5 stop solution: it's 1 mol/L H2SO4

6. Equipment

multichannel pipettes

multichannel photometers

7. and patience

8. start with coating experiments 1 - 10 µg/mL is fine, 100 µL per well also, 50 µL if you have only small amounts of material available, then checkerboard titration's with positive and negative controls to find the optimal working dilution.

Hi,

1. Before developing any type of ELISA you need to study which type of ELISA is best for detection of Ab against Bacteria

2. Because you are detecting Ab against bacteria is very difficult but if you are detecting Ag against specific antigen/s produced by the bacteria is possible.

3. your question is not clear (name of bacteria is not mention)

4. if you can tell us the name then we can suggest the system of detection.

thanks

Hi,

1. Before developing any type of ELISA you need to study which type of ELISA is best for detection of Ab against Bacteria

2. Because you are detecting Ab against bacteria is very difficult but if you are detecting Antibody/es against specific antigen/s produced by the bacteria is possible.

3. your question is not clear (name of bacteria is not mention)

4. if you can tell us the name then we can suggest the system of detection.

thanks

Pasturella multocida

WOW!! Thank you sooo much SWATI!!! Those kits look GREAT! I want to find a better way of identifying PM in birds than by doing my original RT-PCR procedures after swabbing oral and cloacal on birds. I am having a lot of issues with Plasmid contamination and although the test is very sensitive for detecting PM it also seems to read positive for other bacterias which I need to do a lot of gram staining and different broth growth to idenetify if the positives are actually PM bacteria. I was hoping there was an ELISA for detecting the PM antibody in serum that could tell us if the bird has or had avian Cholera. I thought we needed to develop one... this is wonderful news that one is already out there! I did some searches but did not find any. Thank you again!!